Methods,compositions and kits for cell separation

ABSTRACT

Methods, compositions and kits for concentrating or separating cells containing target nucleic acid are disclosed, especially m mixtures containing the cells and other components such as impurities. The methods can keep a large proportion of the cells intact, allowing the cells to be employed after separation (e.g. cultured) and/or which facilitates the recovery of nucleic acid from the cells. The method employs flocculating agents, such as polyamines or cationic detergents, to form complexes with cells causing them to aggregate and so separated from other components of the mixture. Conveniently, the separation of the aggregated cells can be effected with a solid phase which is capable of binding the cells, such as magnetic beads or filters.

FIELD OF THE INVENTION

The present invention relates to methods, compositions and kits for cellseparation, and in particular for separating cells from a mixture inwhich they are present with impurities, and more especially for use inmethods which then allow the purification of target nucleic acid presentin the cells.

BACKGROUND OF THE INVENTION

The separation of cells from mixtures containing them and unwantedimpurities is a challenging problem in the art. This is particularly thecase where the cells are present in a culture broth, a biological sampleor similar complex mixture as the methods employed need to capture ahigh proportion of the cells and capture substantially all of the cellsintact, i.e. without killing or lysing the cells which would cause therelease of cellular debris to further contaminate the mixture. Thismeans that the reagents used in the cell concentration and separationsteps must capture the cells very efficiently and from a range of celldensities, and not interfere by lysing the cell walls or making them“leaky” to nucleic acid before they are separated. Also, the reagentsused should not interfere with downstream steps employing the cells,recovering nucleic acid from the cells and/or the processing of thenucleic acid, e.g. in carrying out PCR or other analytical techniques.

The separation of cells from cultures using flocculating agents such aspolyethylenimine (PEI) is known in the art, see for example Kamath andD'Souza, Enzyme Microb. Technol., 13:935-939, 1991, which reports thecapture of cells on cotton cloth coated with PEI. However, this paper isconcerned with obtaining immobilised cells for use in bioreactors ratherthan the analytical processing of cells or the nucleic acid containedwithin them. Indeed, these prior art methods attempted to remove DNAfrom the cell cultures.

EP 0 515 484 A (Amersham International plc) discloses methods usingmagnetic beads formed from a magnetic material such as iron oxide, andoptionally an organic polymer, for removing impurities such as celldebris, proteins and chromosomal DNA from a lysate mixture, therebyallowing the separation of a supernatant containing nucleic acid ofinterest. This application also discloses the use of the same type ofbeads for precipitating nucleic acid of interest from a supernatant andusing the magnetic properties of the beads to draw down nucleic acidnon-specifically binding to them. In passing, the application alsorefers to the precipitation of bacteria, tissue culture cells and bloodcells using conventional precipitants, such as ethanolic sodium acetateat pH 5.2, and magnetic bead induced precipitate separation. However,the use of alcoholic precipitation in prior art methods suffers from thedisadvantage that it causes cell death and lysis.

WO99/29703 and WO02/48164 (DNA Research Innovations Limited) disclose awide range of ‘charge switch’ materials, typically in the form of solidphases, which are capable of binding nucleic acid present in a sample ata first pH and releasing the nucleic acid at a second, higher pH. Thesecharge switch materials can be employed in the purification of nucleicacid from samples such as biological samples and lysis mixtures. Thematerials can be used in the form of magnetic beads or incorporated onthe surface of pipettes or tubes.

U.S. Pat. No. 6,284,470 (Promega Corporation) discloses kits comprisingtwo species of magnetic beads, a first which forms a complex withdisrupted biological material present in a lysis mixture with a targetnucleic acid and a second which forms a complex with the target nucleicacid under conditions which promote specific adsorption of the nucleicacid to the particles. The second species of magnetic particles may havecharge switch properties, that is the binding of nucleic acid to theparticles is pH dependent. This patent also describes the use ofmagnetic particles to concentrate or harvest cells such as bacteria orwhite blood cells by forming a complex between the cells and magneticbeads, e.g. derivatised with glycidyl-histidine.

There remains a need in the art for new methods of separating cells, andin particular for methods which are largely capable of avoiding celllysis and which are readily susceptible to automation.

SUMMARY OF THE INVENTION

Broadly, the present invention concerns methods, compositions and kitsfor concentrating or separating cells, especially from mixturescontaining the cells and other components such as impurities. Inpreferred aspects, the present invention concerns a method of separatingcells which is capable of keeping a large proportion of the cells intactand which therefore allows the cells to be employed after separation(e.g. cultured) and/or which facilitates the recovery of nucleic acidfrom the cells. The present invention is based on the finding thatflocculating agents, such as polyamines or cationic detergents, formcomplexes with cells causing them to aggregate. For cells present inmixtures, the aggregation of the cells allows them to be readilyseparated from other components of the mixture. Conveniently, theseparation of the aggregated cells can be effected with a solid phasewhich is capable of binding the cells, such as magnetic beads orfilters.

Accordingly, in a first aspect, the present invention provides a methodof separating cells present in a mixture with other materials, themethod comprising:

-   -   contacting the mixture containing the cells with a flocculating        agent capable of aggregating the cells, wherein the flocculating        agent is a polyamine or a cationic detergent, and a solid phase        capable of binding the cells; and,    -   separating the aggregated cells from the mixture using the solid        phase.

The solid phase can be brought into contact with the cells before, afteror simultaneously with the addition of the flocculating agent. In oneembodiment, the flocculating agent is coupled to (preferably covalentlylinked to), mixed with or associated with the solid phase. This has theadvantage of causing the cells to flocculate on the solid phase whichcan then be used to separate the cells from the mixture. In analternative embodiment, the flocculating agent is initially soluble whenadded to the mixture containing the cells and forms an insolubleprecipitate with the cells. In either case, the aggregation orprecipitation of the cells may be enhanced using an agent which promotesor enhances this process as described below.

Examples of suitable solid phases for use in accordance with the presentinvention include magnetic beads, non magnetic beads, filters, filtercolumns, spin filter columns, membranes, particles, beads (e.g. silicabeads) or frits, sinters, glass beads or slides, metal surfaces, fibres,polysaccharides or any plastic surface such as a tube, tip, probe orwell. Magnetic beads are a particularly preferred solid phase,conveniently having average diameters between 0.1-20 μm. The solid phasemay be in a soluble or insoluble form composed of inorganic or organicmaterials or composites thereof. By way of example, the solid phase maycomprise materials such as plastics, glasses, polysaccharides, metaloxides, metal hydroxides/hydrates, salts, silicates, clays, lignins,charcoals and other insoluble fine particulates.

In the present invention, preferably a substantial proportion of thecells are captured intact. This means that the chemicals used mustcapture the cells efficiently, i.e. from a range of cell densities, andnot interfere by killing or lysing the cell walls or making them “leaky”to nucleic acid before they are separated. In preferred embodiments, thepresent invention has the further advantage that the cells are viableafter separation and can therefore be cultured or otherwise employed.Also, it is preferable that the reagents used are compatible withrecovering the nucleic acid from the cells or inhibit downstream nucleicacid analysis, e.g. by PCR or other techniques.

Thus, in the context of the present invention, “not substantially lysed”in the cell separation step of the method means that less than 20%, morepreferably less than 10%, more preferably less than 5%, more preferablyless than 2% and most preferably less than 1% of the cells in thepopulation treated according to the method are lysed. The extent of celllysis can readily be determined, e.g. by counting lysed and non-lysedcells present in a sample under a microscope. As mentioned above, it isalso preferably that a substantial proportion of the cells are viableafter separation according to the present invention. Cell viability canbe readily assessed by growing a sample of the separated cells on anappropriate growth medium and in this context, ‘a substantialproportion’ means at least 50% of the cells are viable, more preferablyat least 75% of the cells, more preferably at least 85% of the cells andmost preferably at least 95% of the cells are viable.

In the present invention, a flocculating agent which is a “polyamine”means a substance having more than one covalently linked units, eachunit having one or more amine groups, e.g. primary, secondary, tertiary,quaternary, aromatic or heterocyclic amine groups, which are positivelycharged at the pH at which the material is used in the cell separationmethod. Preferred polyamines comprise a plurality of covalently linkedunits. The units forming the polyamine may be the same or different. Inaddition to the amine groups, the polyamines may be unsubstituted orsubstituted with one or more further functional groups, e.g. to modifytheir properties of facilitate coupling onto a solid phase. Preferredexamples of polyamines include polyamino acids, polyallylamines,polyalkylimines such as polyethylenimine, polymerised biological bufferscontaining amine groups and polyglucoseamines. All of these classes ofpolyamine may be substituted or unsubstituted. Preferred polyamines, andespecially polyallylamines, have molecular weights in the range of about10 kDa to about 100 kDa, more preferably from about 50 kDa to about 80kDa, and most preferably about 70 kDa. As mentioned above, preferredembodiments of the invention employ polyamines which are initiallysoluble and precipitate on forming complexes with the cells or thepolyamine are coupled to, mixed with or associated with the solid phase.

In embodiments of the invention in which the polyamine is a polyaminoacid, the linked amino acids forming the polyamino acid may be the sameor different. Preferred examples include poly-lysines orpoly-histidines. The amino acids used to form the polyamino acid may beD or L amino acids or a mixture of both.

In embodiments of the invention in which the polyamine is apolyallylamine or polyallylamine. HCl, the polyallylamine is preferablyrepresented by the formula:Poly(allylamine Hydrochloride): [—CH₂CH(CH₂NH₂.HCl)—]_(n) orPoly(allylamine): [—CH₂CH (CH₂NH₂)—]_(n)where n is at least 3 and the polyallylamine may be unsubstituted orhave one or more further substitutions not shown in the simple formulaeabove. Such materials can be produced by the polymerisation of2-propen-1-amine or a similar monomer comprising an alkene and an aminefunctional groups. Examples of polyallylamine can be supplied by Aldrichin the forms of solid of as solutions (e.g. 20 wt % solutions), both ofwhich are usuable according to the present invention. Exemplarypolyallylamines include poly(allylamine) reference 47,914-4 (20 wt %solution, Mw ca 65,000), poly(allylamine) reference 47,913-6 (20 wt %solution, Mw ca 17,000), poly(allylamine hydrochloride) reference28,321-5 (solid, Mw ca 15,000) and poly(allylamine hydrochloride)reference 28,322-3 (solid, Mw ca70,000) all described in the 2001Aldrich Catalogue, page 1385.

In embodiments of the invention in which the polyamine is apolyalkylimines such as polyethylimine (PEI), for example as representedby the formulae: polyethylenimine:(—NHCH₂CH₂—)_(x)[—N(CH₂CH₂NH₂)CH₂CH₂—]_(y).

In embodiments of the invention in which the polyamine is a polymerisedbiological buffer such as poly Bis-Tris. Examples of biological bufferswhich have amine groups and can be polymerised and employed in thepresent invention include:

-   Bis-2-hydroxyethyliminotrishydroxymethylmethane (Bis-Tris), pKa 6.5.-   1,3-bistrishydroxymethylmethylaminopropane (Bis-Tris propane), pKa    6.8.-   N-trishydroxymethylmethylglycine (TRICINE), pKa 8.1.-   Trishydroxymethylaminomethane (TRIS), pKa 8.1.

In embodiments of the invention in which the polyamine is apolyglucoseamine such as chitosan, a readily available material derivedfrom the shells of crustacea and formed from repeating units ofD-glucoseamine.

Other materials useful in flocculating cells are cationic detergents,such as hexamethidrine bromide, benzalkonium chloride, DTAB, CTAB,N-lauryl sarcosine ,cetrimide, polymyxins, or anti-septic oranti-microbial compounds.

In a further aspect, the present invention provides a compositioncomprising a solid phase and a flocculating agent, wherein theflocculating agent is a polyamine or a cationic detergent. As above, theflocculating agent may be associated with, mixed with or coupled to thesolid phase. In embodiments in which the polyamine or detergent iscoupled to the solid phase, covalently coupling is preferred.

In this aspect of the invention, the solid phase is preferably in theform of a bead, and more preferably a magnetic bead, for example havingan average diameter between 0.1-20 μm. The solid phase may be formedfrom a material which is capable of binding nucleic acid at a first pHand releasing the bound nucleic acid at a second higher pH, i.e. acharge switch solid phase, for example as disclosed in WO02/48164 orWO99/29703. This means that one solid phase can be employed in theseparation of cells from impurities and then in the subsequentpurification of nucleic acid contained with the cells. This hasadvantages in simplifying the reagents needed to carry out suchpurification protocols and making them more susceptible to automation.

In a further aspect, the present invention provides a kit for separatingcells from a mixture where the cells are present with impurities, thekit comprising:

-   -   a flocculating agent capable of aggregating the cells, wherein        the flocculating agent is a polyamine or a cationic detergent;    -   a first solid phase which is capable of binding the aggregated        cells;    -   optionally a second solid phase for purifying nucleic acid in        the cells, the solid phase being capable of binding nucleic acid        at a first pH and releasing the bound nucleic acid at a second        higher pH (i.e. a charge switch solid phase, for example as        disclosed in WO02/48164 or WO99/29703).

In preferred kits, the first and second solid phases may be the same,i.e. a charge switch solid phase can be employed to bind the cells andalso in the purification of nucleic acid contained with the cells.

The present invention is widely applicable to many different types ofsamples containing cells including, but not limited to, culture broths,biological samples such as blood and tissue, foodstuffs, watercontaminated liquids, host cells, e.g. separating cells such as Gramnegative and Gram positive bacteria (e.g. E. coli), filamentous bacteriaor fungi (such as Streptomyces), yeast cells, mammalian cells, plantcells and plant protoplasts.

In some preferred embodiments of the invention, the flocculating agentis used in conjunction with an agent to promote the aggregation of thecells. This agent may be a change in pH or temperature, a divalent orpolyvalent ion, a change in counter ion to the flocculating agent, across-linking agent, a change-in concentration, evaporation. In apreferred embodiment of the invention, divalent or polyvalent anions areadded to the mixture containing cells in order to promote flocculation.In a particularly preferred embodiment, phosphate ions are added.However, the phosphate ions may be substituted for any divalent orpolyvalent anion including, but not limited to, sulphates andpolycarboxylates. Without wishing to be bound by any particular theory,the inventors believe that when divalent cations such as phosphates areused, a polyelectrolyte complex is formed that becomes insoluble aroundthe cells aiding the aggregation of cells and hence separation.

To carry out cell separation, the cell sample is brought in contact withthe flocculating agent and solid phase. The cells associate with them,allowing the solid phase to be used to remove the complex from solution.Separation may be achieved by a range of well, known in the art such asvacuum filtration, syringe filtration, magnetic separation,electrophoresis, centrifugation, sedimentation or evaporation or liquidremoval techniques.

After separation, the cells may be collected and cultured, stored forarchive purposes or treated to release important biomolecules such asnucleic acids, proteins, metabolites, carbohydrates or lipid componentsor complexes thereof. Significant lysis of the cells during separationis avoided so that the biomolecules inside the cell are not lost. Thus,in a further embodiment, the methods of the present invention maycomprise the step of culturing cells separated from the mixture.

The method of separating cells may be followed with steps to purifytarget biomolecules, and especially nucleic acid, contained within thecells. By way of example, the target nucleic acid may be non-genomicnucleic acid which is separated from genomic nucleic acid retainedinside the cells. Non-genomic nucleic acid includes vectors, plasmids,self replicating satellite nucleic acid or cosmid DNA, or vector RNA.Other forms of target nucleic acids may include bacteriophages such asLambda, M13 and viral nucleic acids. In a preferred embodiment, thenon-genomic nucleic acid sample is plasmid DNA.

In preferred embodiments, the method is used to separate cellscontaining nucleic acid of interest, and the initial step of aggregatingthe cells may be part of a method of purifying the nucleic acid, asdescribed in more detail below. Thus, in such embodiments of theinvention, the method may comprise additional processing or purificationsteps carried out on the cell sample, for example involving one or moreof the additional steps of:

-   -   (a) isolating the target nucleic acid; or    -   (b) analysing the target nucleic acid; or    -   (c) amplifying the target nucleic acid; or    -   (d) sequencing the target nucleic acid.

These steps are discussed in more detail below.

In a preferred embodiment, the invention may further comprise obtaininga sample of target nucleic acid from cells containing the target nucleicacid and genomic nucleic acid, the method comprising having separatedthe cells from culture broth, the further steps of:

-   -   suspending the cells in an aqueous medium which causes the        target nucleic acid to leak from the cells into the aqueous        medium; and    -   obtaining the sample of the nucleic acid from the aqueous        medium;    -   wherein the cells are substantially not lysed during the above        steps and substantially retain the genomic nucleic acid within        the cells.

The details of this method are provided in PCT/GB02/005209. Preferably,this method does not substantially cause the release of cellularendotoxins, thereby allowing the separation of the target nucleic acidfrom the cellular endotoxins, in addition to genomic nucleic acid orRNA. In a preferred embodiment of this method, the target nucleic acidsmay be 100 kb or less, or more preferably 50 kb or less, or morepreferably 20 kb or less or even more preferably 10 kb or less in size.The size of nucleic acids can be determined by those skilled in the art,e.g. using gel electrophoresis technique employing a polyacrylamide oragarose gel, e.g. see Ausubel et al, Short Protocols in MolecularBiology, John Wiley and Sons, NY, 1992.

Alternatively, the cells separated according to the above method may belysed and target nucleic acid purified from the lysate, for exampleusing a charge switch solid phase referred to above, a nucleic acidbinding solid phase as described in EP 0 389 063 A in which silica or aderivative thereof is used to bind nucleic acid in the presence of achaotrope.

In either case, the target nucleic acid, such as a plasmid, can beseparated from the media containing the cells according to the presentinvention and the resulting aqueous media, i.e. the supernatant, useddirectly with out the requirement for further purification steps, e.g.for PCR or other analytical methods.

A range of techniques are available to the skilled person for purifyingnucleic acid are known in the art. Examples of purification techniquesinclude ion-exchange, electrophoresis, silica solid phase withchaotropic salt extraction, precipitation, flocculation, filtration, gelfiltration, centrifugation, alcohol precipitation and/or the use of acharge switch material described in our copending applicationsWO97/29703 and WO02/48164 and other purification or separation methodswell known in the art. In preferred embodiments, the target nucleic acidis purified using a charge switch material, e.g. present on a solidphase, a pipette tip, beads (especially magnetic beads), a porousmembrane, a frit, a sinter, a probe or dipstick, a tube (PCR tube,Eppendorf tube) or a microarray.

The target nucleic acid may also be the subject of amplification,conveniently using the polymerase chain reaction. PCR techniques for theamplification of nucleic acid are described in U.S. Pat. No. 4,683,195.In general, such techniques require that sequence information from theends of the target sequence is known to allow suitable forward andreverse oligonucleotide primers to be designed to be identical orsimilar to the polynucleotide sequence that is the target for theamplification. PCR comprises steps of denaturation of template nucleicacid (if double-stranded), annealing of primer to target, andpolymerisation. The nucleic acid probed or used as template in theamplification reaction may be genomic DNA, cDNA or RNA. PCR can be usedto amplify specific sequences from genomic DNA, specific RNA sequencesand cDNA transcribed from mRNA, bacteriophage or plasmid sequences.References for the general use of PCR techniques include Mullis et al,Cold Spring Harbor Symp. Quant. Biol., 51:263, (1987), Ehrlich (ed), PCRTechnology, Stockton Press, NY, 1989, Ehrlich et al, Science,252:1643-1650, (1991), “PCR protocols; A Guide to Methods andApplications”, Eds. Innis et al, Academic Press, New York, (1990).

Embodiments of the present invention will now be described in moredetail by way of example and not limitation.

DETAILED DESCRIPTION EXAMPLE 1 Polyamine Flocculation, Capturing Cellson a Filter and Purifying DNA Using Charge Switch Magnetic Beads

0.75 ml of an overnight culture of E. coli/pUC19 was mixed with 10 μl of50 mg/ml poly(allylamine hydrochloride), Mw=70 kDa approx., in a 0.45 μmspin-filter column for 1 minute. The spin-filter column was thencentrifuged at 13000 rpm for 1 minute to remove liquid without blockingthe filter and the flow through was discarded. In the spin-filtercolumn, the pellet was resuspended in 100 μl of 10 mM Tris-HCl (pH 8.5),1 mM EDTA buffer containing 100 μg/ml RNaseA and left for 1 minute. Theresuspended cells were then mixed with 100 μl of a 1% (w/v) SDS, 0.2 MNaOH lysis solution for 3 minutes, then a precipitation buffer (1.0 Mpotassium acetate, 0.66 M KCl, pH 4.0) was gently mixed in toprecipitate cell debris. The spin-filter column was centrifuged againfor 1 minute at 13000 rpm and the flow through was mixed with 20 μl ofCST magnetic beads (25 mg/ml) and incubated at room temperature for 1minute. Samples were applied to a magnet for 1 min and the supernatantwas discarded. The beads were then washed twice with 100 μl of distilledwater and then purified plasmid DNA was eluted from the beads into 50 μlof 10 mM Tris-HCl (pH8.5). Purified plasmid DNA was visualised by gelelectrophoresis in a 1% agarose gel containing ethidium bromide.

EXAMPLE 2 Polyamine Flocculation, Capturing Cells on Charge SwitchMagnetic Beads and Purifying DNA Using Charge Switch Magnetic Beads

1.0 ml of an overnight culture of E. coli/pUC19 was mixed with 30 μl ofCST magnetic beads (25 mg/ml) premixed with 5 mg/ml poly(allylaminehydrochloride), Mw=70 kDa approx., in a 1.5 ml microcentrifuge tube for1 minute. The sample was then applied to a magnet for 1 minute toharvest the magnetic beads and flocculated cells. The supernatant wasdiscarded and the magnetic pellet was resuspended in 100 μl of 10 mMTris-HCl (pH 8.5), 1 mM EDTA buffer containing 100 μg/ml RNaseA and leftfor 1 minute. The resuspended cells were then mixed with 100 μl of a 1%(w/v) SDS, 0.2 M NaOH lysis solution for 3 minutes, then a precipitationbuffer (1.0 M potassium acetate, 0.66M KCl, pH 4.0) was gently mixed into precipitate cell debris. Cell debris was removed by applying thesample to a magnet for 1 minute. The supernatant was then mixed with 20μl of CST magnetic beads (25 mg/ml) and incubated at room temperaturefor 1 minute. Samples were applied to a magnet for 1 minute and thesupernatant was discarded. The beads were then washed twice with 100 μlof distilled water and then purified plasmid DNA was eluted from thebeads into 50 μl of 10 mM Tris-HCl (pH8.5). Purified plasmid DNA wasvisualised by gel electrophoresis in a 1% agarose electrophoresis gelcontaining ethidium bromide.

EXAMPLE 3 Polyamine Flocculation, Capturing Cells on Particles ofMagnetite and Purifying DNA Using Charge Switch Magnetic Beads

As example 2, but using 50 μl of magnetite (50 mg/ml) premixed with 10mg/ml poly(allylamine hydrochloride), Mw=70 kDa approx., instead of 30μl of CST magnetic beads (25 mg/ml) premixed with 5 mg/mlpoly(allylamine hydrochloride), Mw=70 kDa approx.

EXAMPLE 4

As example 2, but using 10 mg/ml poly(allylamine hydrochloride), Mw=70kDa approx., instead of 5 mg/ml poly(allylamine hydrochloride), Mw=70kDa approx.

EXAMPLE 5

As example 2, but using 10 mg/ml poly-L-lysine instead of 5 mg/mlpoly(allylamine hydrochloride), Mw=70 kDa approx.

EXAMPLE 6

As example 2, but using 10 mg/ml poly-DL-lysine instead of 5 mg/mlpoly(allylamine hydrochloride), Mw=70 kDa approx.

EXAMPLE 7

As example 2, but using 10 mg/ml poly-L-histidine instead of 5 mg/mlpoly(allylamine hydrochloride), Mw=70 kDa approx.

EXAMPLE 8

As example 2, but using 10 mg/ml poly(allylamine hydrochloride), Mw=15kDa approx., instead of 5 mg/ml poly(allylamine hydrochloride), Mw=70kDa approx.

EXAMPLE 9

As example 2, but using 1 mg/ml poly(allylamine), Mw=17 kDa approx.,instead of 5 mg/ml poly(allylamine hydrochloride), Mw=70 kDa approx.

EXAMPLE 10

As example 2, but using 1 mg/ml poly(allylamine), Mw=65 kDa approx.,instead of 5 mg/ml poly(allylamine hydrochloride), Mw=70 kDa approx.

EXAMPLE 11

As example 2, but using 10 mg/ml poly(ethylenimine), instead of 5 mg/mlpoly(allylamine hydrochloride), Mw=70 kDa approx.

EXAMPLE 12

As example 2, but using 10 mg/ml polymyxin B (Sigma-Aldrich cataloguenumber P-1004), instead of 5 mg/ml poly(allylamine hydrochloride), Mw=70kDa approx.

EXAMPLE 13

As example 2, but using 10 mg/ml benzalkonium chloride, instead of 5mg/ml poly(allylamine hydrochloride), Mw=70 kDa approx.

EXAMPLE 14

As example 2, but using 10 mg/ml hexadecytrimethylammonium bromide(‘Cetrimide’, ‘CTAB’) instead of 5 mg/ml poly(allylamine hydrochloride),Mw=70 kDa approx.

EXAMPLE 15 Mammalian Cell Separation

Red blood cell (RBC) lysis solution=10 mM NH4HCO3, 0.1% Tween 20.

White blood cell (WBC) digestion buffer=1% SDS, 1 mM EDTA, 10 mM TrisHCl pH8

Genomic precipitation buffer=6 M ammonium acetate.

10 ml of sheep's blood was mixed with 30 ml of ‘RBC lysis solution’ andincubated at room temperature for 10 minutes. The sample was thencentrifuged at 2000 rpm for 10 min and the supernatant was discarded andthe cell pellet was resuspended in 10 ml of 50 mM phosphate buffer. A500 μl aliquot of the cell suspension was then mixed with 30 μl of CSTmagnetic beads (25 mg/ml), premixed with 1 mg/ml poly(allylaminehydrochloride), Mw=70 kDa approx., and incubated for 2 minutes. Thesample was then held against a magnet for 2 minutes and thecell-suspension was seen to be clear, indicating that the cells had beenremoved from suspension. The supernatant was discarded and the pelletwas resuspended in 500 μl of ‘WBC digestion buffer’ and mixed bypipetting up and down for 1 minute. 150 μl of ‘Genomic precipitationbuffer’ was then added and the mixture was vortexed for 20 seconds, theresulting precipitate was removed by applying the sample to a magnet for2 minutes. 500 μl of the supernatant was then gently mixed with 500 μlof isopropanol and genomic DNA was seen to form a precipitate.

EXAMPLE 16

As example 15, but using 1 mg/ml poly-L-lysine instead of 1 mg/mlpoly(allylamine hydrochloride), Mw=70 kDa approx.

EXAMPLE 17

As example 15, but using 1 mg/ml poly-DL-lysine instead of 1 mg/mlpoly(allylamine hydrochloride), Mw=70 kDa approx.

EXAMPLE 18

As example 15, but using 1 mg/ml poly-L-histidine instead of 1 mg/mlpoly(allylamine hydrochloride), Mw=70 kDa approx.

EXAMPLE 19

As example 15, but using 1 mg/ml poly(allylamine hydrochloride), Mw=15kDa approx., instead of 1 mg/ml poly(allylamine hydrochloride), Mw=70kDa approx.

EXAMPLE 20

As example 15, but using 1 mg/ml poly(allylamine), Mw=17 kDa approx.,instead of 1 mg/ml poly(allylamine hydrochloride), Mw=70 kDa approx.

EXAMPLE 21

As example 15, but using 1 mg/ml poly(allylamine), Mw=65 kDa approx.,instead of 1 mg/ml poly(allylamine hydrochloride), Mw=70 kDa approx.

EXAMPLE 22

As example 15, but using 1 mg/ml poly(ethylenimine), instead of 1 mg/mlpoly(allylamine hydrochloride), Mw=70 kDa approx.

EXAMPLE 23

As example 15, but using 1 mg/ml polymyxin B (Sigma-Aldrich cat. No.P-1004), instead of 1 mg/ml poly(allylamine hydrochloride), Mw=70 kDaapprox.

EXAMPLE 24

As example 15, but using 1 mg/ml benzalkonium chloride, instead of 1mg/ml poly(allylamine hydrochloride), Mw=70 kDa approx.

EXAMPLE 25

As example 15, but using 1 mg/ml ‘Cetrimide’ (hexadecyltrimethylammoniumbromide) instead of 1 mg/ml poly(allylamine hydrochloride), Mw=70 kDaapprox.

EXAMPLE 26

As example 15, but omitting 5 mg/ml poly(allylamine hydrochloride),Mw=70 kDa approx., and using only the poly-Tris coated magnetic beads

EXAMPLE 27

200 μl of sheep's blood was mixed with 600 μl ‘RBC lysis solution’ andincubated at room temperature for 10 minutes. The sample was then mixedwith 50 μl of CST magnetic beads (25 mg/ml), premixed with 1 mg/mlpoly(allylamine hydrochloride), Mw=70 kDa approx., and incubated for 2minutes. The sample was then applied to a magnet for 2 minutes and thesupernatant was discarded. The magnetic pellet was resuspended in 200 μlof 10 mM NaOH and incubated at room temperature for 1 minute. Theresuspended pellet was then held against a magnet for 2 min to removemagnetic particles. Extracted DNA was then visualised by gelelectrophoresis in a 1% agarose gel containing ethidium bromide.

EXAMPLE 28

As example 27, but using 1 mg/ml poly-DL-lysine instead of 1 mg/mlpoly(allylamine hydrochloride), Mw=70 kDa approx.

EXAMPLE 29

As example 27, but using 1 mg/ml poly-L-histidine instead of 1 mg/mlpoly(allylamine hydrochloride), Mw=70 kDa approx.

EXAMPLE 30

As example 27, but using 1 mg/ml poly(allylamine hydrochloride), Mw=15kDa approx., instead of 1 mg/ml poly(allylamine hydrochloride), Mw=70kDa approx.

EXAMPLE 31

As example 27, but using 1 mg/ml poly(allylamine), Mw=17 kDa approx.,instead of 1 mg/ml poly(allylamine hydrochloride), Mw=70 kDa approx.

EXAMPLE 32

As example 27, but using 1 mg/ml poly(allylamine), Mw=65 kDa approx.,instead of 1 mg/ml poly(allylamine hydrochloride), Mw=70 kDa approx.

EXAMPLE 33

As example 27, but using 1 mg/ml poly(ethylenimine), instead of 1 mg/mlpoly(allylamine hydrochloride), Mw=70 kDa approx.

EXAMPLE 34

As example 27, but using 1 mg/ml polymyxin B (Sigma-Aldrich cataloguenumber P-1004), instead of 1 mg/ml poly(allylamine hydrochloride), Mw=70kDa approx.

EXAMPLE 35

As example 27, but using 1 mg/ml benzalkonium chloride, instead of 1mg/ml poly(allylamine hydrochloride), Mw =70 kDa approx.

EXAMPLE 36

As example 27, but using 1 mg/ml ‘Cetrimide’ (hexadecytrimethylammoniumbromide) instead of 1 mg/ml poly(allylamine hydrochloride), Mw=70 kDaapprox.

EXAMPLE 37

As example 27, but omitting 5 mg/ml poly(allylamine hydrochloride),Mw=70 kDa approx. and using only the poly-Tris coated magnetic beads.

EXAMPLE 38

200 μl of sheep's blood was mixed with 600 μl ‘RBC lysis solution’ andincubated at room temperature for 10 minutes. The sample was then mixedwith 50 μl of CST magnetic beads (25 mg/ml), premixed with 1 mg/mlpoly(allylamine), Mw=65 kDa approx., and incubated for 2 minutes. Thesample was then applied to a magnet for 2 min and the supernatant wasdiscarded. The magnetic pellet was resuspended in 500 μl of ‘WBCdigestion buffer’ and mixed by pipetting for 1 minute. 150 μl of‘Genomic precipitation buffer’ was added and vortexed for 20 seconds tomix then the tube was placed against a magnet for 2 minutes. 500 μl ofthe supernatant was removed and mixed with 500 μl of isopropanol toprecipitate any DNA. The sample was then incubated at −20° C. for 2 minfollowed by centrifugation at 13000 rpm for 10 minutes. The supernatantwas discarded and the pellet was washed once with 500 μl of 70% (v/v)ethanol. The pellet was air-dried and then dissolved overnight in 10 mMTris-HCl. The purified genomic DNA was then visualised by gelelectrophoresis in a 1% agarose gel containing ethidium bromide.

EXAMPLE 39

As example 38, but using 1 mg/ml poly-L-lysine instead of 1 mg/mlpoly(allylamine hydrochloride), Mw=70 kDa approx.

EXAMPLE 40

As example 38, but using 1 mg/ml poly-DL-lysine instead of 1 mg/mlpoly(allylamine hydrochloride), Mw=70 kDa approx.

EXAMPLE 41

As example 38, but using 1 mg/ml poly-L-histidine instead of 1 mg/mlpoly(allylamine hydrochloride), Mw=70 kDa approx.

EXAMPLE 42

As example 38, but using 1 mg/ml poly(allylamine hydrochloride), Mw=15kDa approx., instead of 1 mg/ml poly(allylamine hydrochloride), Mw=70kDa approx.

EXAMPLE 43

As example 38, but using 1 mg/ml poly(allylamine), Mw=17 kDa approx.,instead of 1 mg/ml poly(allylamine hydrochloride), Mw=70 kDa approx.

EXAMPLE 44

As example 38, but using 1 mg/ml poly(allylamine), Mw=65 kDa approx.,instead of 1 mg/ml poly(allylamine hydrochloride), Mw=70 kDa approx.

EXAMPLE 45

As example 38, but using 1 mg/ml poly(ethylenimine), instead of 1 mg/mlpoly(allylamine hydrochloride), Mw=70 kDa approx.

EXAMPLE 46

As example 38, but using 1 mg/ml polymyxin B (Sigma Aldrich cataloguenumber P-1004), instead of 1 mg/ml poly(allylamine hydrochloride), Mw=70kDa approx.

EXAMPLE 47

As example 38, but using 1 mg/ml benzalkonium chloride, instead of 1mg/ml poly(allylamine hydrochloride), Mw=70 kDa approx.

EXAMPLE 48

As example 38, but using 1 mg/ml ‘Cetrimide’ (hexadecytrimethylammoniumbromide) instead of 1 mg/ml poly(allylamine hydrochloride), Mw=70 kDaapprox.

EXAMPLE 49

As example 38, but omitting 5 mg/ml poly(allylamine hydrochloride),Mw=70 kDa approx. and using only the poly-Tris coated magnetic beads

EXAMPLE 50 Cells Separated Using the Present Invention can be Cultured

1 ml of overnight culture of E. coli/pUC19 was mixed with 30 μl of 50mg/ml poly(allylamine hydrochloride), Mw=70 kDa approx. The resultingflock formed from the precipitation reaction was removed from the brothwith a sterile inoculation loop and streaked out on to LBA platescontaining 50 μg/ml ampicillin (to select for the β-lactamase gene onthe pUC19 plasmid). Plates were incubated overnight at 37° C. Goodbacterial growth was seen, indicating that the flocculation reaction didnot kill the bacteria.

EXAMPLE 51

1 ml of overnight culture of E. coli/pUC19 was mixed with 30 μl of CSTbeads premixed with 5 mg/ml poly(allylamine hydrochloride), Mw=70 kDaapprox. The resulting magnetic precipitate was harvested by holding thetube against a magnet for 1 minute and discarding the supernatant. Thepellet was then streaked on to LBA plates containing 50 μg/ml ampicillin(to select for the β-lactamase gene on the pUC19 plasmid) using asterile inoculation loop. Plates were then incubated overnight at 37° C.Good bacterial growth was seen, indicating that the flocculationreaction did not kill the bacteria.

EXAMPLE 52

Plasmid DNA purified using the method described in example 2 can bedigested using restriction endonucleases (such as HindIII), showing thatDNA can be used in molecular biological applications.

EXAMPLE 53

1.0 ml of an overnight culture of E. coli/pUC19 was mixed with 50 μl ofmagnetite (50 mg/ml) premixed with 1 mg/ml Chitosan in a 1.5 mlmicrocentrifuge tube for 1 minute. The sample was then applied to amagnet for 1 minute to harvest the magnetic beads and flocculated cells.The supernatant was discarded and the magnetic pellet was resuspended in100 μl of 10 mM Tris-HCl (pH 8.5), 1 mM EDTA buffer containing 100 μg/mlRNaseA and left for 1 minute. The resuspended cells were then mixed with100 μl of a 1% (w/v) SDS, 0.2M NaOH lysis solution for 3 minutes, then aprecipitation buffer (1.0M potassium acetate, 0.66M KCl, pH 4.0) wasgently mixed in to precipitate cell debris. Cell debris was removed byapplying the sample to a magnet for 1 minute. The supernatant was thenmixed with 20 μl of CST magnetic beads (25 mg/ml) and incubated at roomtemperature for 1 minute. Samples were applied to a magnet for 1 minuteand the supernatant was discarded. The beads were then washed twice with100 μl of distilled water and then purified plasmid DNA was eluted fromthe beads into 50 μl of 10 mM Tris-HCl (pH8.5). Purified plasmid DNA wasvisualised by gel electrophoresis in a 1% agarose electrophoresis gelcontaining ethidium bromide.

EXAMPLE 54 Purification of Yeast Vectors

An overnight culture of yeast YPH501 containing vector ESC-Leu wasprepared and 1 ml was mixed with 30 μl of magnetic beads adsorbed withpolyamine. After the cells were separated with a magnet the supernatantwas removed and the cells resuspended in a standard spheroplastingsolution containing sorbital, mercaptoethanol and lyticase for 30minutes. The spheroplasts were then lysed with 300 ul of 0.2M NaOH with1% SDS which was then cleared by adding 30 ul of a 1.5M potassiumacetate buffer pH4. Removal of the cellular debris was achieved by usingthe magnetic beads still present in the mixture to bind to the debrisand separate with a magnet.

The references herein all expressly incorporated by reference.

1. A method of separating cells containing target nucleic acid, thecells being present in a mixture with other materials, and of purifyingthe target nucleic acid from the cells, the method comprising: (a)contacting the mixture containing the cells with a flocculating agentcapable of aggregating the cells, wherein the flocculating agent is apolyamine or a cationic detergent, and a solid phase capable of bindingthe cells; (b) separating the aggregated cells from the mixture, usingthe solid phase; and (c) purifying the target nucleic acid from thecells.
 2. The method of claim 1, wherein the solid phase is brought intocontact with the cells before, after or simultaneously with the additionof the flocculating agent.
 3. The method of claim 1 or claim 2, whereinthe cells are not substantially lysed after the separation step.
 4. Themethod of any one of claims 1 to 3, wherein the cells are viable afterthe separation step.
 5. The method of any one of the preceding claims,wherein the flocculating agent is coupled to, mixed with or associatedwith the solid phase causing the cells to flocculate on the solid phasewhich can then be used to remove the cells from the mixture.
 6. Themethod of any one of claims 1 to 5, wherein the flocculating agent isinitially soluble and forms a precipitate with the cells in the mixture.7. The method of any one of the preceding claims, wherein the solidphase comprises magnetic beads, non magnetic beads, filters, membranes,particles, silica beads or frits, sinters, glass, polysaccharides or anyplastic surface such as a tube, tip, probe or well.
 8. The method ofclaim 7, wherein the solid phase is magnetic beads.
 9. The method of anyone of the preceding claims, wherein the solid phase is capable ofbinding nucleic acid at a first pH and releasing nucleic acid at asecond, higher pH.
 10. The method of any one of the preceding claims,further comprising adding divalent or polyvalent anions to the mixtureto promote flocculation of the cells.
 11. The method of claim 10,wherein the divalent or polyvalent anions are phosphate or sulphateions.
 12. The method of claim 10 or claim 11, wherein the anions areadded before or after the flocculating agent.
 13. The method of any oneof the preceding claims, wherein flocculating agent is a polyamine. 14.The method of claim 13, wherein the polyamine is a polyamino acid, apolyallylamine, a polyalkylimine, a polyethylimine, a polymerisedbiological buffer containing amine groups, or a polyglucoseamine. 15.The method of any one of claims 1 to 12, wherein flocculating agent is acationic detergent
 16. The method of claim 15, wherein the cationicdetergent is hexamethidrine bromide, benzalkonium chloride, DTAB, CTAB,N-lauryl sarcosine, cetrimide, polymyxins, or an anti-septic oranti-microbial compound.
 17. The method of any one of the precedingclaims, wherein the cells are present in. a culture broth or abiological sample.
 18. The method of any one of the preceding claims,further comprising culturing the cells after separation from themixture.
 19. The method of any one of the preceding claims, whereinafter step (b) the cells are lysed.
 20. The method of claim 19, furthercomprising, after the step of lysing the cells, the step of binding celldebris to the solid phase and separating the cell debris and solid phaseto provide a solution of target nucleic acid.
 21. The method of claim20, further comprising separating the nucleic acid from the solution.22. The method of claim 21, wherein the nucleic acid is separated usinga solid phase comprising silica or a derivative thereof to bind thenucleic acid.
 23. The method of claim 21, wherein the nucleic acid isseparated by contacting the solution of target nucleic acid with a solidphase is capable of binding nucleic acid at a first pH and releasingnucleic acid at a second, higher pH so that the nucleic acid binds tothe solid phase.
 24. The method of claim 23, further comprising changingthe pH of the solution to the second, higher pH to release the targetnucleic acid.
 25. The method of any one of the preceding claims, furthercomprising analysing and/or amplifying and/or sequencing the targetnucleic acid.
 26. The method of any one of claims 1 to 18, furthercomprising: obtaining a sample of target nucleic acid from cellscontaining the target nucleic acid and genomic nucleic acid, the methodcomprising having separated the cells from culture broth, the furthersteps of: suspending the cells in an aqueous medium which causes thetarget nucleic acid to leak from the cells into the aqueous medium; andobtaining the sample of the nucleic acid from the aqueous medium;wherein the cells are substantially not lysed during the above steps andsubstantially retain the genomic nucleic acid within the cells.
 27. Acomposition comprising a solid phase mixed with a flocculating agent,wherein: (a) the flocculating agent is a polyamine or a cationicdetergent; and (b) the solid phase is a magnetic bead or the solid phaseis formed from a material which is capable of binding nucleic acid at afirst pH and releasing the bound nucleic acid at a second higher pH. 28.A kit for separating cells from a mixture where the cells are presentwith impurities and purifying nucleic acid present in the cell, the kitcomprising: a flocculating agent capable of aggregating the cells,wherein the flocculating agent is a polyamine or a cationic detergent; afirst solid phase which is capable of binding the aggregated cells; anda second solid phase for purifying nucleic acid in the cells, the solidphase being capable of binding nucleic acid at a first pH and releasingthe bound nucleic acid at a second higher pH.
 29. The kit of claim 28,wherein the first and second solid phases are the same.
 30. The kit ofclaim 28 or claim 29, wherein the first and/or the second solid phasesare beads.
 31. The kit of claim 30, wherein the bead is a magnetic bead.32. The kit of any one of claims 28 to 31, wherein the solid phase isformed from a material which is capable of binding nucleic acid at afirst pH and releasing the bound nucleic acid at a second higher pH.